G-quadruplex (G4) is a noncanonical secondary structure of DNA or RNA which can enhance or repress gene expression, yet the underlying molecular mechanism remains uncertain. Here we show that when positioned downstream of transcription start site, the orientation of potential G4 forming sequence (PQS), but not the sequence alters transcriptional output. Ensemble in vitro transcription assays indicate that PQS in the non-template increases mRNA production rate and yield. Using sequential single molecule detection stages, we demonstrate that while binding and initiation of T7 RNA polymerase is unchanged, the efficiency of elongation and the final mRNA output is higher when PQS is in the non-template. Strikingly, the enhanced elongation arises from the transcription-induced R-loop formation, which in turn generates G4 structure in the non-template. The G4 stabilized R-loop leads to increased transcription by a mechanism involving successive rounds of R-loop formation.

G-四链体 (G4) 是 DNA 或 RNA 的非规范二级结构，可以增强或抑制基因表达，但潜在的分子机制仍不确定。在这里，我们表明，当位于转录起始位点的下游时，潜在 G4 形成序列 (PQS) 的方向会改变转录输出，但不会改变序列。Ensemble 体外转录分析表明非模板中的 PQS 提高了 mRNA 的生产速率和产量。使用连续的单分子检测阶段，我们证明虽然 T7 RNA 聚合酶的结合和起始不变，但当 PQS 在非模板中时，延伸效率和最终 mRNA 输出更高。引人注目的是，增强的延伸源于转录诱导的 R 环形成，进而在非模板中产生 G4 结构。